Cloning, expression, purification and kinetics of trehalose-6-phosphate phosphatase of filarial parasite Brugia malayi

• Published in: Acta tropica Vol. 119; no. 2-3; pp. 151 - 159
• Main Authors: Kushwaha, Susheela, Singh, Prashant K., Rana, Ajay K., Misra-Bhattacharya, Shailja
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier B.V 01.08.2011; Elsevier
• Subjects: Animals; Antifilarial; Biological and medical sciences; biosynthesis; Brugia malayi; Brugia malayi - enzymology; Brugia malayi - genetics; Caenorhabditis elegans; Cloning, Molecular; Coenzymes - metabolism; Diseases caused by nematodes; Drug target; drugs; Enzyme Stability; enzymes; Filariases; filariasis; Gene Expression; General aspects; Helminthic diseases; Hydrogen-Ion Concentration; Infectious diseases; Kinetics; Lymphatic filariases; Lymphatic filariasis; magnesium; Magnesium - metabolism; mammals; Medical sciences; Nematoda; parasites; Parasitic diseases; Phosphoric Monoester Hydrolases - chemistry; Phosphoric Monoester Hydrolases - genetics; Phosphoric Monoester Hydrolases - isolation & purification; Phosphoric Monoester Hydrolases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Substrate Specificity; Sugar Phosphates - metabolism; trehalose; Trehalose - analogs & derivatives; Trehalose - metabolism; Trehalose-6-phosphate phosphatase; Drug target; Antifilarial; Trehalose-6-phosphate phosphatase; Brugia malayi; Lymphatic filariasis; Purification; Enzyme; Phosphoric monoester hydrolases; Parasite; Cardiovascular disease; Esterases; Parasitosis; Helminthiasis; Lymphatic vessel disease; Infection; Nematode disease; Helmintha; Hydrolases; Tropical medicine; Nemathelminthia; Kinetics; Invertebrata; Nematoda; Trehalose-phosphatase
• ISSN: 0001-706X; 1873-6254
• DOI: 10.1016/j.actatropica.2011.05.008

Trehalose-6-phosphate phosphatase from B. malayi was cloned, expressed and purified. Its unusual phosphatase activity and absence in mammals demonstrated its worth as biochemical drug target. [Display omitted] ► Bm-TPP was cloned, expressed and found to possess robust phosphatase activity. ► It shows specificity towards trehalose-6-phosphate and belongs to HAD super family. ► The enzyme shows absolute requirement for Mg2+ as a metal ion for activity. The pleiotropic functions of disaccharide trehalose in the biology of nematodes and its absence from mammalian cells suggest that its biosynthesis may provide a useful target for developing novel nematicidal drugs. The trehalose-6-phosphate phosphatase (TPP), one of the enzymes of trehalose metabolism has not been characterized so far in nematodes except the free living nematode Caenorhabditis elegans where it's silencing results into lethal outcomes. This prompted us to clone and characterize Brugia malayi TPP in order to discover novel antifilarial drug target. The recombinant protein (Bm-TPP) was purified with apparent homogeneity on a metal ion column and it was found to possess high phosphatase activity with robust specificity for the substrate trehalose-6-phosphate. Bm-TPP was found to be a member of the HAD-like hydrolase super family II based on the conserved motifs required for catalytic reaction. The Km for substrate trehalose-6-phosphate was around 0.42mM with pH optimum ∼7.0 and the enzyme showed an almost absolute requirement for Mg2+ as a metal ion. Bm-TPP was expressed in all the life-stages of B. malayi. In the absence of an effective macrofilaricidal agent and validated antifilarial drug target, Bm-TPP bodes well as a rational drug target against lymphatic filariasis.